Milk oligosaccharides (MOS) and galactooligosaccharides (GOS) are associated with many benefits, including anti-microbial effects and immune-modulating properties. However, the cellular mechanisms of these are largely unknown. In this study, the effects of enriched GOS and MOS mixtures from caprine and bovine milk consisting mainly 6'-galactosyllactose, 3'-sialyllactose, and 6'-sialyllactose on Caco-2 cells were investigated, and the treatment-specific metabolomes were described. In the control, the cells were treated with a sugar mix consisting of one-third each of glucose, galactose and lactose.A local metabolomics workflow with pathway enrichment was established, which specifically addresses DI-FT-ICR-MS analyses and includes adaptations in terms of measurement technology and sample matrices. By including quality parameters, especially the isotope pattern, we increased the precision of annotation. The independence from online tools, the fast adaptability to changes in databases, and the specific adjustment to the measurement technology and biomaterial used, proved to be a great advantage.For the first time it was possible to find 71 active pathways in a Caco-2 cell experiment. These pathways were assigned to 12 main categories, with amino acid metabolism and carbohydrate metabolism being the most dominant categories in terms of the number of metabolites and metabolic pathways. Treatment of Caco-2 cells with high GOS and glucose contents resulted in significant effects on several metabolic pathways, whereas the MOS containing treatments resulted only for individual metabolites in significant changes. An effect based on bovine or caprine origin alone could not be observed. Thus, it was shown that MOS and GOS containing treatments can exert microbiome-independent effects on the metabolome of Caco-2 cells. These include prebiotic, anti-adhesive, anti-microbial, and immune-modulating effects . In addio models , 2017. To models . MOS areo models . Caprineo models . GOS, aso models . Since to models . In the o models . Howevero models . The maio models . Howeverin vitro system to study the interaction between intestinal cells and oligosaccharides are Caco-2 cells. Caco-2 cell lines are a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells , e.g., with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), is able to cover a large range of metabolites in a short measurement time, even for complex sample matrices without prior chromatographic separation. In addition, it has extremely high resolution (xHR) in comparison to various LC-MS analytical techniques and very high sensitivity in comparison to NMR . In partThe first aim of the study was to establish a metabolomics workflow which improves the identification level for DI-FT-ICR-MS analyses, which is usually referred to the lowest level G  or 1 as All solvents listed were obtained from Carl Roth, Karlsruhe, Germany. All consumables were purchased from Sarstedt, Nümbrecht, Germany.K. lactis. The high GOS (HG) fraction contained different mono-, di-, tri- and tetrasaccharides, with 6'-galactosyllactose as main product. Bovine and caprine skimmed ultrafiltrated (UF) milks were subjected to nanofiltration. For better separation of MOS from residual sugars, lactose in the UF-permeate was hydrolyzed by ß-galactosidase. High GOS/bMOS (GM) from cow’s milk fraction contained the bovine MOS N-acetyl-β-D-galactosamine-lactose, 6'-sialyllactose, and 3'-sialyllactose as main products. Low GOS/cMOS (LGM) from goat’s milk contained 6'-sialyllactose and 3'-sialyllactose as main component of the caprine MOS fraction beside other minor MOS. Both fractions contained in addition comparable amounts of GOS, monosaccharides and lactose from the lactose hydrolysis during sample preparation and further milk components like milk salts. Control (C) consisted of glucose, galactose, and lactose , as they were also part of the GOS and MOS fractions. All GOS/MOS contents were determined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). A detailed overview of the oligosaccharides compositions is summarized in Oligosaccharides were produced according to −1]; Biochrome AG, Berlin, Germany) at 37°C and 5% CO2. For all experiments cells were seeded at a density of 1 × 105–2×105 cells cm−2 to ensure confluency after O/N incubation. Medium was changed every second day. After 20 days, cells were treated in accordance with the experiments of −1  between passage 24 and 50 were cultured in T-175 polystyrene flasks  with Caco-2 medium . The upper phase was transferred to a new tube and kept at 4°C. A second extraction cycle was conducted as described above. The resulting upper lipophilic phase and middle hydrophilic phase were transferred into 15 ml tubes, and the lower proteinogenic phase was discarded. The phases were dried with a speedvac and resuspended with the following liquids: The hydrophilic compounds (middle phase) were resuspended in methanol/water  with 0.1% acetic acid (500 µL). The lipophilic compounds (upper phase) were resuspended in isopropanol/chloroform  with 0.1% acetic acid (500 µL). Samples were stored at −80°C until measurement. This work focused on the investigation of the hydrophilic compounds.Cells were removed from the culture flasks under standardized conditions using a cell scraper. Thereafter, 3 times 2 ml of ultra-pure water with 0.1% (v/v) formic acid was added and treated for 2 min each. Cell disruption was conducted by ultrasonication  on ice. The cell lysate was dried by means of a speedvac  and resuspended in 1.5 ml ultra-pure water. Extraction was performed using a modified SIMPLEX approach based on measurementvia FT-ICR-MS (A), peak alignment (B) was conducted using MetaboScape 4.0 SR1 . For data analysis and metabolite annotation (C) in the first step (C1.1), an organism-specific database was selected and downloaded n”] were not considered for further analysis. In addition, only sufficiently large (≥60Da) and thus potentially measurable compounds were considered. Drugs and associated pathways were also excluded. The resulting, cleaned local database was then used for metabolite annotation in MetaboScape. Thereafter, the theoretical masses were calculated based on the sum formulas and compared and matched with the measured masses (C2.1). The resulting preliminary annotations were again subjected to filtering according to quality parameters (C2.2). These quality parameters included the mass error and the isotope pattern (see 2.2.5). Confirmed formulas and thus confirmed IDs of tentative metabolites were merged into a new export file, which in turn was used to map KEGG compounds to pathways in which the compounds participate (C3.1). The resulting pathway information and associated metabolites were used for pathway enrichment analysis. To evaluate and filter the enrichments, the following quality parameters were considered (C3.2): percentage of coverage, enrichment factor, occurrence of pathway-specific sum formulas, and number of linked metabolites (see 2.2.7).The workflow for the metabolomics analyses is presented in ormulas; ). The refeature statistics were performed and included principal component analysis (PCA) as well as differential feature statistics to identify differences in feature intensities between treatments. In the final step (E), the results of the workflow were compared with two different web-databases.(D) In parallel, non-targeted comparisons MassTRIX  were used. Further settings for MassTRIX: database KEGG, HMDB, Lipidmaps without isotopes, organism Homo sapiens. For the comparative web-annotation, the data resulting from Step B were used. The measured masses were exported as a bucket table . From the web-based comparisons, only metabolites for which a KEGG ID was available were used for further analyses. Based on this data, a new local database was created for MetaboScape, which also passed through workflow steps C1.1 to C3.1.For the Germany; ) and HMDGermany; ) were usFor the mass spectrometric analysis, a FT-ICR-MS system  equipped with an ESI source was used. For the direct injection (DI), an Infinity 1260 HPLC autosampler and pump  was linked. The eluent consisted of methanol and water  with 0.1% acetic acid. For analyses samples were ionized in positive or negative modes by electro spray ionization. The mass range was between 60 and 1,500 m/z. The main parameters were: Nitrogen as dry gas (4 L/min and 200°C) and nitrogen as nebulizer (1 bar); the time-of-flight section was set to 0.35 ms; the quadrupole mass was set to 200 m/z; the quadrupole RF was 2 MHz and the sweep excitation power of the ICR-cell was 18%. All methods were calibrated using sodium trifluoroacetate. The stability of the measurement was monitored using quality control samples (QC). The QC represents a pool of all samples measured every 20th analysis .FT-ICR-MS data were processed by MetaboScape 4.0 . All datasets were recalibrated by matrix compounds within the samples  with a tolerated error <0.5 Da. To reduce noise, only peaks were selected which were present in at least 20 of 96 samples. Further, the intensity threshold was set to 500,000 counts.hsa, and to the measurement technique. Peaks were annotated if the mass error was below 2 ppm and if the mSigma (isotope error) was below 300 . The annotation was considered individually for each ion: [M + H]+, [M + Na]+, and [M + K]+ (positive mode), and [M-H]− and [M + Cl]− (negative mode), respectively. The annotated data were exported as tab-separated tables of the feature intensities for subsequent statistical analysis in R version 4.1.1. If a metabolite was present in the form of several ions, the ion with the highest intensity count was used for the statistical analysis.For metabolite annotation, the database resulting from the workflow was used with adaptations to the organism, i.e., p-values that describe the significance of different feature intensities between treatments were obtained by likelihood-ratio tests of the full model (with treatment as a fixed effect) against the reduced model without treatment type as a variability-explaining factor. Median fold changes  How many metabolites of a pathway were found in the metabolomics data relative to the number of metabolites in the same pathway that could theoretically be measured using the FT-ICR-analysis if above the LOD? 2) Which pathways are enriched in metabolites that have been identified to be significantly abundant, dependent on the treatment type based on pairwise-treatment comparisons using a linear mixed effect model statistic (see 2.2.6)? The metabolites ATP, ADP, AMP, NAD, NADH, NADP, NADPH, coenzyme A, phosphate, and diphosphate were not considered in the pathway enrichment analysis as these metabolites participate in a wide range of diverse pathways and are therefore not informative for the activity of specific pathways. Furthermore, only metabolites that are theoretically measurable by the FT-ICR-MS approach were included in the pathway enrichment analysis, which excludes metabolites with a mass of less than 60 Da and metabolites with ambiguous sum formula . Enrichment factors of significant treatment-dependent metabolites for each pathway (HG), a combination with a high content of GOS/bMOS (GM), a combination with a low GOS/cMOS content (LGM), and a control (C) which consisted of one-third each of glucose, galactose, and lactose. Since the treatments were not pure substances, and also contained e.g., mono- and disaccharides, the different treatments were compared in six combinations.• Each individual treatment vs. control• The two GOS/MOS treatments against each other• The two GOS/MOS treatments individually against the high GOS treatmentIn a semi-targeted metabolomics approach, the bovine and caprine milk- (b- and cMOS) and galactooligosaccharide (GOS) treatment-specific Caco-2 metabolomes should be investigated. For this purpose, four different sugar combinations were used: a high content of GOS The multi-comparisons allow us to distinguish whether differences between metabolomics profiles are due to the monosaccharides, the disaccharides, or the presence of GOS or MOS. Moreover, general and concentration-dependent effects could be inferred .(A), peak alignment (B), data analysis (C), and statistics (D). Element E evaluates comparability with workflows using web-based approaches with databases or online tools. A key objective of the presented workflow is the reduction of false-positive results at the level of metabolites and pathways through specific filtering steps using various quality parameters (see 2.2.5/2.2.7).A local workflow has been developed and applied, which specifically addresses the highly complex metabolomics data from DI-FT-ICR-MS analyses. This workflow includes five basic elements: Measurement Based on this workflow we identified all metabolites and associated pathways characterizing the Caco-2 metabolome and treatment-specific effects using different sugar mixtures. The numeric results of the individual steps of the metabolomics workflow are shown in via MetaboScape considering the quality parameters.Peak alignment resulted in 5,945 features (B), a feature is defined as the measured mass (m/z) without any annotation. Based on the annotation list obtained after the first filter step (C.1.2), containing all potentially measurable metabolites and sum formulas, 211 sum formulas with 468 corresponding KEGG IDS were assigned to the 5,945 features. This corresponds to a percentage share of 3.6%. Matching of metabolites to the corresponding metabolic pathways by KEGG mapping resulted in 83 preliminary metabolic pathways with at least 3 hits (C3.1); metabolites listed in 2.2.7 were excluded. Based on the specified quality parameters of the pathway enrichment analyses, a further 12 of 83 pathways were excluded (C3.2). Multiple comparisons in the statistical analyses revealed that 25 of the remaining 71 metabolic pathways were significantly regulated (D). Step E shows the difference between annotation with web-based approaches and the results validated Pathway enrichment analyses were used to reduce the number of false-positive annotations by applying various quality criteria (see 2.2.5/2.2.7). These included a pathway coverage of at least 33.33%, the occurrence of at least three directly linked non-isobaric metabolites within one pathway, and the occurrence of unique sum formulas.versus those sum formulas which have been determined, a coverage rate of 22.86% is expected across all 83 pathways. 46 pathways were above the expected value, eight of them significantly . Applying the quality parameters resulted in 35 pathways passing the 33.33% level, 42 containing at least one unique molecular formula and, 28 having at least 3 directly linked metabolites. In the following the percentage distribution of the 83 pathways based on the number of fulfilled criteria is listed:• 5 of 83 pathways fulfilled 3 criteria, corresponding to 6.02%• 24 of 83 pathways fulfilled 2 criteria, corresponding to 28.92%• 42 of 83 pathways fulfilled 1 criterion, corresponding to 50.60%• 12 of 83 pathways did not fit any of the criteria, corresponding to 14.46%Pathway mapping based on the annotated metabolites resulted in 83 preliminary pathways having a minimum of 3 hits. Based on all sum formulas that could potentially be determined 12 pathways did not meet any of the criteria and were thus excluded from further analyses. For detailed information see All 71 remaining identified pathways, which fulfilled at least one criterion, were assigned to 24 different main categories based on the KEGG nomenclature , i.e., a percentage coverage of 48.83%, two metabolites with unique sum formulas, and more than 3 linked metabolites. Thus, the pathway is considered to be confirmed. Both metabolites fructose-6-phosphate and glucose-6-phosphate are present in this pathway and directly linked to each other, i.e., fructose-6-phosphate is derived from glucose-6-phosphate, and subsequent metabolites such as fructose-1,6-bisphosphate, sedoheptulose-7-phosphate, and 2-deoxy-ribose phosphates have also been detected. Therefore, the co-occurrence of both isobars is plausible.Furthermore, based on ascorbate and aldarate metabolism, it was possible to exclude the arabinonates, since neither precursor molecules nor subsequent metabolites were detected. But the presence of isobaric compounds xylonates and lyxonates is proposed, since two precursor molecules were detected. Ascorbate and aldarate metabolism fulfilled all 3 criteria with a coverage rate of 42.31%Based on pyrimidine metabolism, the presence of the isobars glutamine and 3-ureidoisobutyrate would be excluded, as they appear without directly linked metabolites in the pathway. However, glutamine can be confirmed by alanine, aspartate, and glutamate metabolism, as it is directly linked to two further metabolites in this pathway. Pyrimidine metabolism is (with 24.56%) below the minimum percentage coverage but fulfills the other two criteria. Alanine, aspartate, and glutamate metabolism fulfills only the minimum criterion of linked non-isobaric metabolites.To visualize overall similarities and differences in the cell metabolome caused by the treatments, and to give a general overview, a PCA was used. The metabolomes of the control, GM, and LGM exhibited similar scattering patterns in the PCA, whereas HG showed clear clustering in PC1 vs. PC2 and PC2 vs. PC3 in the score plots . In accop -value < 0.05). These pathways are categorized in Across all previously described comparisons, there were a total of 25 significantly regulated pathways . Regulation of 2-isopropylmaleate, 1-aminopropan-2-ol-O-phosphate, and 2-amino-3-phosphonopropanoate was found for a high GOS amount (HG vs. C/GM/LGM) and of 3-dehydro-threonate for HG vs. C. Creatine was significantly regulated in the context of the MOS fraction (GM/LGM vs. HG and GM vs. C) and regulation of shikimate-3-phosphate, 3-hydroxy-2-methylpyridine-4,5-dicarboxylate, ribosyl-homocysteine, and 1-myo-inositol-1,4,5 triphosphate was identified for GM (GM vs. HG/C).The first aim of the study was to establish a local metabolomics workflow which improves the identification level for DI-FT-ICR-MS analyses, and to investigate the presence of isobaric compounds.The second aim was to characterize the Caco-2 metabolome in detail, objectively applying the new workflow and investigating how milk oligosaccharide (MOS) and galactooligosaccharide (GOS) containing sugar mixtures influence the metabolome of Caco-2 cells.Using the presented workflow, we were able to identify 71 pathways, 202 sum formulas, and 445 KEGG IDs based on 5,945 features measured. Comparing these numbers with the web-based annotations using HMDB (397 sum formulas and 735 KEEG IDs) or the web-based tool MassTRIX (766 sum formulas and 1857 KEGG IDs) the number of identified metabolites were lower in our workflow. The differences can be explained by our filtering steps, which included quality parameters that evaluated isotopic patterns and fine structures (see 2.2.5), thus leading to a higher precision in identification. Further, pathway enrichment analyses were used as filtering elements which resulted in a lower number of confirmed annotations. Further reduction was caused by the exclusion of drug metabolism pathways, and metabolites with non-defined residues or with non-specified number of repetitions for structural elements in our study. Nevertheless, our results are plausible and comparable with online based workflows using HMDB and MassTRIX. After entering workflow step C2.1/C2.2 with the resulting sum formulas from HMDB or MassTRIX, followed by pathway mapping, all pathways identified in our workflow were covered. Repeating the evaluation step C2.1/C2.2 was necessary because the web databases only use mass error for identification, while local processing in our workflow also considers the isotopic pattern, and thus reduces the number of false-positive annotations see . A compaAnother important quality parameter to reduce the number of false annotations, was pathway coverage, which was calculated based on sum formulas. Here we followed the conservative procedure according to Further, evaluating the co-existence of isobaric compounds was improved for the first time using pathway enrichment analyses. This step allowed us to analyze the plausibility of metabolite annotations by integrating them into a pathway and directly linking metabolites to the biological context. Thus, in the pentose phosphate pathway, the co-existence of the two isobars fructose-6-phosphate and glucose-6-phosphate was verified. In addition, the occurrence of 3-ureidoisobutyrate could be excluded, whereas the occurrence of the isobaric compound glutamine was confirmed by alanine, aspartate, and glutamate metabolism. However, there are also limitations; terminal isobaric compounds of a pathway with the same precursor molecules cannot be further evaluated for their presence without additional analysis. The same applies to the occurrence of isobaric compounds with precursor molecules and degradation products that are also isobaric compounds.In vivo, intestinal epithelial cells are the interface between host and microbiome and are exposed to many microenvironmental metabolites  and fructose and mannose metabolism (C vs. HG). Caco-2 cells are able to transfer glucose from the surrounding medium into the cell via the GLUT1 or SGLT1 transporters  by contributing to the regeneration of oxidized glutathione (We showed a significant regulation of pentose phosphate metabolism (C/LGM vssporters . The p-vment see . It is ktathione . An incrtathione . This covia transcytosis (A significantly higher content of tri- and disaccharides was observed in the HG-treated cells (HG vs. C/GM/LGM). 6'-galactosyllactose is a neutral oligosaccharide, which can enter Caco-2 cells by transcellular transport scytosis , 2002. Ivia glutaminase, which is in turn metabolized to alpha-ketoglutarate and supplied to the citrate cycle, resulting in malate. Malate was also detected in the HG-treated cells with significantly higher content compared to the control. Malate can cytosolically be degraded to pyruvate or, alternatively, via oxaloacetate, to aspartate and via this contribute to asparagine regeneration and nucleotide synthesis , including glutamate, glutamine, aspartate, and asparagine. An increase in glutamate in Caco-2 cells in the context of inulin treatment was also described by ynthesis . The amiynthesis . Since v+ symporters. It can be assumed that there is an excess of Na+ ions in the cell in connection with the increased uptake of amino acids. Taurine contributes to preventing this imbalance.The significantly higher amino acid content in the cells treated with HG may also be due to increased transport activity, as all amino acids were also part of the medium. Significantly higher levels of histidine and glycerol-3-phosphates were detected in all treatments containing GOS. This could be related to increased proton shuttle processes. The glycerol-3-phosphate shuttle system contributes to the transport of cytosolically produced NADH into the mitochondria, which is subsequently transferred to the respiratory chain for ATP production. The inner mitochondrial membrane is impermeable to NADH, so that a shuttle-dependent transport must take place . Histidivia paracellular transport in Caco-2 monolayers. As described by Significantly higher levels of the second messenger 1-myo-inositol-1,4,5-triphosphate (IP3) were observed in GM-treated cells (GM vs. C/HG/LGM). Thus, the present response must be based on cell surface sensing. The regulation could be related to the sialyllactose present in the bMOS fraction. Sialyllactose is an acidic oligosaccharide due to its side chain. The established local workflow offers an alternative to web-based approaches. It considers isotope pattern as well as mass error, additional quality parameters that lead to higher precision in identification and annotation. By incorporating the biological context through the pathway enrichment analyses and the criteria established for the enrichment analyses, it is possible to evaluate the co-presence of isobars. A further advantage of the local workflow is its independence and fast adaptability. The entire workflow can be applied to other cell culture systems and organisms as well as to various treatments and measurement techniques, whereby isotope fine structure can only be used as a quality parameter for xHR instruments.Applying the presented workflow allows a detailed characterization of the Caco-2 metabolome and forms the basis for further investigations of bioactive compounds in cell culture experiments.Moreover, it was shown for the first time that GOS and MOS can directly, i.e., independently of the microbiome, affect the metabolome of Caco-2 cells, ranging from single metabolites to the regulation of entire pathways. Most importantly amino acid pathways (D-glutamine and D-glutamate metabolism and histidine metabolism) were upregulated by GOS vs. the other treatments.


This study focused on characterizing the volatile profiles and contributing compounds in pan-fried steaks from different Chinese yellow cattle breeds. The volatile organic compounds (VOCs) of six Chinese yellow cattle breeds  were analyzed by GC-Q-Orbitrap spectrometry and electronic nose (E-nose). Multivariate statistical analysis was performed to identify the differences in VOCs profiles among breeds. The relationship between odor-active volatiles and sensory evaluation was analyzed by partial least square regression (PLSR) to identify contributing volatiles in pan-fried steaks of Chinese yellow cattle. The results showed that samples were divided into two groups, and 18 VOCs were selected as potential markers for the differentiation of the two groups by GC-Q-Orbitrap combined multivariate statistical analysis. YL and WS were in one group comprising mainly aliphatic compounds, while the rest were in the other group with more cyclic compounds. Steaks from different breeds were better differentiated by GC-Q-Orbitrap in combination with chemometrics than by E-nose. Six highly predictive compounds were selected, including 3-methyl-butanal, benzeneacetaldehyde, 2-ethyl-6-methyl-pyrazine, 2-acetylpyrrole, 2-acetylthiazole, and 2-acetyl-2-thiazoline. Sensory recombination difference and preference testing revealed that the addition of highly predictive compounds induced a perceptible difference to panelists. This study provides valuable data to characterize and discriminate the flavor profiles in pan-fried steaks of Chinese yellow cattle. China has been breeding yellow cattle for a long time and exhibits diverse yellow cattle resources. According to the Chinese Catalogue for Livestock and Poultry Genetic Resources , there aBeef flavor is mainly associated with numerous volatile organic compounds (VOCs). More than 800 beef VOCs have been identified , includiVOCs in beef are commonly analyzed by gas chromatography (GC) coupled with triple quadrupole mass spectrometer (MS), but this approach is limited by low resolution . In ordeIn this study, strip loin steaks from six yellow cattle breeds that are commonly available on the market were selected. Volatile profiles of pan-fried steaks from these cattle breeds were determined by GC-Q-Orbitrap and E-nose combined with multivariate data analysis. Partial least squares regression (PLSR) analysis between VOCs and sensory evaluation was carried out to obtain volatiles highly predictive of the flavor of Chinese yellow cattle. Sensory recombination difference and preference testing were performed to verify whether these highly predictive compounds contributed significantly to the beef flavor.Qualitative and quantitative analyses of VOCs in pan-fried steaks from different yellow cattle breeds are listed in p < 0.05). These three compounds potentially result from the Strecker degradation of leucine, tyrosine, and phenylalanine, respectively [Aldehydes represented a large group of VOCs found in the pan-fried steaks. As shown in ectively ,26. Mostectively . Benzaldectively . 3-Methyectively . p < 0.05). These two compounds played important roles in roast beef [N-, O-, and S-containing compounds were abundant in Chinese yellow cattle and exhibited distinct odor characteristics. PL samples were the most abundant in pyrazines, which are mainly formed by the condensation of carbonyl ammonia compounds generated by the Strecker degradation reaction ,29. Pyraast beef . Thiazolast beef . The conast beef . 2-Pentyast beef . Two furast beef . Althougast beef . The prep < 0.05). Alcohols have high odor thresholds and are produced from lipid oxidation or carbonyl compound reduction [p < 0.05). Acetoin was considered an important contributor to the buttery odor in beef [Most alcohols in WS were significantly higher than those in other breeds  and predicted efficiently (Q2 = 0.966). The OPLS-DA model was validated by cross-validation ANOVA (p < 0.05), indicating the model fitted well. In addition, the importance of various substances was evaluated by variable importance prediction (VIP). VOCs with a VIP score of >1 and p < 0.05 were considered as potential markers for the discrimination of different groups [According to the classification results of PCA and HCA, supervised OPLS-DA was then performed to verify the validity of clustering and find the key markers to discriminate Group 1 and Group 2. The OPLS-DA score plot a showed t groups . As showt groups . Frank et groups . GorraizA p < 0.0, indicatt groups . We infeE-nose was also used to analyze the flavor profiles based on the sensitivity ranges of the sensors. The radar chart shows how the mean responses of the sensors differed with the samples a. BH hadFurthermore, PCA was performed to investigate the differences between the six breeds. As shown in the PCA score plot b, PC1 anBoth GC-Q-Orbitrap and E-nose, in combination with chemometrics, were effective methods to discriminate the volatile profiles of pan-fried steaks from different Chinese yellow cattle breeds. However, the results were not exactly the same. This may be attributed to the comprehensive and fuzzy analysis of E-nose, resulting in limited access to information, or to the low sensitivity of the E-nose sensors to certain substances, leading to the omittance of these substances. Li et al. (2018) explored the correspondence between the responses of E-nose sensors and VOCs measured by GC-MS, and the result showed that only a portion of volatiles could be captured by the sensors . Bai et p < 0.05). The result of aroma scores was the same as that of PCA based on GC-Q-Orbitrap determination. The categories and contents of VOCs had a great effect on the intensity of flavor that people perceived. The contribution of VOCs to flavor was not only related to the content but also to their odor thresholds and interactions, as only specific VOCs with aroma activities could be perceived by people. These odor-active volatiles are the ones that contribute to flavor. To figure out odor-active volatiles (OAVs), the ratio of VOCs concentration to the threshold were calculated. The odor-active volatiles were defined as the VOCs with OAVs ≥ 1 [The scores of beef aroma intensities are shown in OAVs ≥ 1 . As show2 was up to 87.2%, and the permutation test showed that the intercept of the regression line through Q2 was negative, indicating that the model fitted well. 2, was greater than 0.7, indicating good linearity. p < 0.05). 3-Methyl-butanal , benzeneacetaldehyde (honey/sweet odor), 2-ethyl-6-methyl-pyrazine (roasted odor), 2-acetylpyrrole (caramel/fatty/nutty odor), 2-acetylthiazole (grainy/nutty odor), and 2-acetyl-2-thiazoline (popcorn-like odor) may exert a significant positive influence on the flavor attribute. To further estimate the contribution of odor-active volatiles to the flavor of Chinese yellow cattle comprehensively, PLSR analysis between the contents of odor-active volatiles measured by GC-Q-Orbitrap and aroma scores was performed. As shown in The select compounds predictive of aroma intensity were found to be present at lower levels in WS (the lowest score of aroma intensity) compared to PL (the highest score of aroma intensity) . The WS The result confirmed that six highly predictive compounds played important roles in Chinese yellow cattle. This was in accordance with the result of MacLeod G et al. , who rep6–C30) were purchased from Sigma-Aldrich Chemical Co. . 2-Methyl-3-heptanone standard was purchased from Dr. Ehrenstorfer GmbH Co. . 2-Ethyl-6-methyl-pyrazine (97.2%) and 2-acetylpyrrole (99.8%) were obtained from Tan-Mo Technology Co. . 3-Methyl-butanal (95%), benzeneacetaldehyde (95%), 2-acetylthiazole (99%), and 2-acetyl-2-thiazoline (97%) were obtained from Aladdin Biochemical Technology Co. . Propylene glycol (food-grade) was purchased from Guangzhou Yehu Chemical Co. .Methanol (HPLC-grade) and n-alkanes standard  from six Chinese yellow cattle breeds, including Bohai Black Cattle (BH), Jiaxian Red Cattle (JX), Yiling Cattle (YL), Wenshan Cattle (WS), Xinjiang Brown Cattle (XJ), and Pingliang Red Cattle (PL) were used in this experiment. All cattle were grain-fed steers and slaughtered at 36 ± 2.4 months of age with the same commercial slaughter processing. Strip loins of Chinese yellow cattle were vacuum packed and aged at 4 °C for 14 days after slaughter. They were then frozen at −20 °C and transported to the lab at the Institute of Animal Sciences of the Chinese Academy of Agricultural Sciences . The strip loins were immediately sliced into 15 mm thick steaks perpendicular to the direction of the muscle fibers and individually vacuum-packed and stored at −20 °C. After thawing at 4 °C for 2 h, steaks were fried on the pan (200 °C) until their central temperature reached 67 ± 2 °C. Then four slices of each sample were quickly minced, ground with liquid nitrogen, divided into cryopreservation tubes, and stored at −80 °C for GC-Q-Orbitrap, E-nose analysis, and difference and preference testing for recombination. The rested steaks were used for the evaluation of aroma intensity. 6–C30). The NIST 2.3 and WILEY 9 libraries were used to identify compounds with a positive and negative similarity index greater than 800. The peak areas of the target compound and the internal standard were used for quantitative analysis.Four grams of minced sample were accurately weighed and placed in a sealed glass vial (20 mL). 2-methyl-3-heptanone  was added as the internal standard. Samples were then equilibrated at 60 °C for 10 min and determined using a GC-Q-Orbitrap system  consisting of a multi-purpose autosampler, a trace 1310 GC, an electron ionization (EI) source, and a Q-Orbitrap mass spectrometer. The solid-phase microextraction with a DVB/CAR/PDMS SPME fiber was used as the autosampler. The fiber was inserted into the headspace of the vial, and the extraction was operated at 60 °C for 30 min. The fiber was then automatically injected into a GC-Q-Orbitrap equipped with a DB-Wax capillary column (30 m × 250 μm × 0.25 μm)  and was desorbed at 250 °C for 3 min with splitless mode and aged at the same temperature for 20 min. The initial temperature of the GC oven was set at 40 °C for 2 min. The temperature was then raised to 230 °C at a rate of 4 °C/min and maintained for 5 min (run time: 54 min). High-purity helium (99.99% purity) was used as carrier gas and flowed at 1.0 mL/min. Samples were then analyzed with an MS detector in FullScan mode. Electron impact (EI) was used as the ion source, and the ionization energy was 70 eV. The ionization temperature was 280 °C. The quantity scanning range was from 30 to 400. Nitrogen gas (99.99% purity) was used for the C-Trap supply. X-Calibur and Trace Finder 5.1 software  were used for peak identification. Meanwhile, the retention index (RI) was calculated according to the retention times of the n-alkanes standard  , with son = 18) were evaluated in each test session. Evaluating in triplicate required three sessions. An orientation sample from an extra steak was evaluated, and the scores were given by the panel discussion at one time before each session. Unsalted crackers and drinking water were served to avoid the cross-link effects between samples [Sensory evaluation was conducted in the standard sensory evaluation lab at room temperature (20–23 °C). A panel consisting of 17 panelists  assessed the aroma intensity. Panelists were trained according to the procedure of Dikeman et al. (2013)  before s samples .A total of 599.4 g of minced WS sample spiked with 600 µL of propylene glycol was used as the control group for the difference and preference testing. A stock solution of propylene glycol and highly predictive compounds  were added to 599.4 g of WS sample, and the mixture was used as the recombination group. The concentrations of highly predictive compounds were determined by the difference between their concentrations in the most intense sample and the WS sample . Four grA triangle test was performed to determine if there was a difference between the control and recombination groups. Sixty panelists  who reported consuming steaks greater than once per month were recruited from the Institute of Animal Sciences of the CAAS. Two samples from the control group and one sample from the recombination group or one sample from the control group and two samples from the recombination group were presented to panelists in a randomized order. Panelists were asked to select which sample was different from the other two samples by nose smelling and tasting. In addition, one sample from the control group and one sample from the recombination group were presented to panelists. Panelists were asked to choose their preferred sample. Testing was also performed in the standard sensory evaluation lab at room temperature (20–23 °C). Unsalted crackers and drinking water were used as a palate cleanser between samples.p < 0.05). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were performed using MetaboAnalyst 5.0 online. Orthogonal partial least squares-discriminant analysis (OPLS-DA) and PLSR analysis were performed using SIMCA software .One-way analysis of variance (ANOVA) was carried out using SPSS software . The significance of data was compared using Duncan’s multiple range test (Ninety-two VOCs of pan-fried steaks from six Chinese yellow cattle breeds were identified by GC-Q-Orbitrap, and aldehydes were the most abundant components. Six cattle breeds could be divided into two groups by GC-Q-Orbitrap combined with chemometrics. Eighteen VOCs were selected as potential markers for the differentiation of the two groups, and they were mostly the degradation products of lipids. The YL and WS group comprised mainly aliphatic compounds, while most substances in the BH, JX, XJ, and PL group were cyclic compounds. E-nose, in combination with chemometrics, was also an effective method to discriminate the volatile profiles of different breeds, but the results were not exactly the same with GC-Q-Orbitrap due to the different recognition mechanisms for volatiles. PLSR analysis of sensory evaluation and odor-active volatiles identified 3-methylbutanal, benzeneacetaldehyde, 2-ethyl-6-methyl-pyrazine, 2-acetylpyrrole, 2-acetylthiazole, and 2-acetyl-2-thiazoline as key volatiles that affected the flavor attributes of Chinese yellow cattle. Sensory recombination difference and preference testing confirmed that these six highly predictive compounds contributed significantly to the beef aroma. In further study, GC combined with olfactometry technology can be utilized to explore the role of individual flavor component. In addition, further study can be carried out to find the precursors that produce these key volatiles. This can provide directions for nutritional regulation and breeding elite germplasm.


Treatment of metastasized or recurrent oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinoma remains challenging. Targeted antibody-based therapy inter alia for PD-1 / PD-L1 axis shows promising results, but whether PD-L1 expression varies between the subentities remains unclear. The expression pattern of PD-L1  and p16  was determined immunohistochemically and analyzed by HALO™ Image Analysis Software . For PD-L1, combined positivity score (CPS), tumor proportion score (TPS) and histoscore, were assessed and results correlated with epidemiological data. In total, 161 patients  were included. A mean of 43.6% (±34.0%) of the specimen showed increased PD-L1 expression that did not differ quantitatively between subentities , but qualitatively (histoscore: p = 0.003). In the mean follow-up period (45.6 months), contrary to age (p = 0.006) and advanced T-Status (p = 0.018), PD-L1 expression did not correlate with overall  and recurrence free survival (RFS: p = 0.193) in both subentities. No correlation of p16 and PD-L1 expression was found (p = 0.844). PD-L1 is differentially expressed between OSCC and OPSCC, however without influence on OS. Furthermore, p16 status was not related to PD-L1 expression. This may have implications for future (immune) therapeutical approaches for oral cancer. Head and neck squamous cell carcinoma (HNSCC) and its subentities oral (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) represent a life threatening disease that causes around 160,000 deaths each year , 3. AbouIn this context, inhibition of checkpoint pathways of carcinogenesis has provided a new and promising approach in the direction of personalized medicine: Programmed cell death-1 (PD-1), an important inhibitory receptor, is critical for the maintenance of central and peripheral T cell tolerance . HoweverPatients with surgically treated OSCC or OPSCC (n = 161) of the Department of Otorhinolaryngology and the Department of Oral and Maxillofacial Surgery – Plastic Surgery, University Medical Centre, Mainz Germany, were included in the study. Informed consent was obtained from all individuals participated in the study. The analysis was performed within the Declaration of Helsinki and its later amendments and the approval of the local ethic committee . All participants gave informed consent.th Edition), tumor localization, OS, RFS, PD-L1 expression and HPV p16 status were analyzed for all patients. Distant metastasis at the time point of initial staging that led to a primarily systemic therapy was defined as exclusion criteria.All patients received primary surgical tumor therapy with or without adjuvant therapy in accordance with recommendation of the interdisciplinary tumor conference recommendation and existing guidelines. Age, gender, risk behavior, TNM classification  of the respective primary tumors as previously described . BrieflyThe expression of the HPV surrogate marker protein p16 was examined immunohistochemically as previously described , 22. HerThe stained tumor tissue samples were evaluated blindly by a pathologist using the HALO™ Image Analysis Software . Combined Positivity Score (CPS) and Tumor Proportion Score (TPS) as quantitative analyses of expression were achieved. The CPS is defined as all (PD-L1)-stained cells / viable tumor cells x 100 . The TPSStatistical analysis was performed using SPSS version 23 . Results were displayed in mean with its standard error of mean (SEM). Kaplan-Meier graphs with log rank test and cox regression analysis were performed to analyze OS and RFS and possible associations. Differences between the groups were compared using the Mann-Whitney U test since non normal distribution was detected via Shapiro-Wilk-Test. Pearson’s chi-square was used to examine correlations between variables . As conventional, a p-value ≤ 0.05 was set to be significant.A total of 161 patients were included in the study . The meaOf the 161 patients in this study, 73 had died within the median follow-up of 45 months (47.4%). The Kaplan-Meier analysis showed overall no difference for OS between OSCC and OPSCC (p = 0.448). Log Rank test found advanced T status as an independent predictor for reduced OS in both entities . However, in qualitative assessment, OSCC displayed a significant higher histoscore in comparison to OPSCC  displayed a positive p16 expression. In comparison to OSCC, statistically significant more OPSCC were found p16 positive . Within the OPSCC collective, subanalysis showed that tumors of the palatine tonsil were significantly more often associated with a positive p16-status than any other anatomical localization (p = 0.012). Overall, there was no association between the HPV + or HPV- OPSCC and OSCC and PD-L1 expression pattern . A positive p16 status had a significant impact on OS of OPSCC (p = 0.016) and tented to influence RFS (p = 0.032) in comparison to HPV- OPSCC. For OSCC, no further association to OS and/or RFS could be found.The aim of this study was to analyze PD-L1 expression pattern of OSCC and OPSCC comparatively and to detect possible correlations with p16 status as well as epidemiological features such as overall- und recurrence free survival. In the current literature, the PD-L1 expression revealed a wide range and reached from the lowest percentage of 9.5%  to the hIn addition, major drawback of quantitative analysis of PD-L1 expression consists in the fact that an optimal cutoff seems not clear yet to distinguish if an immune-therapeutically approach may be feasible. This becomes all the more important as immune therapy can cause causes a variety of immune-related adverse events that may ultimately attribute to patients’ death .The rating of PD-L1-postivity in the current studies followed either a binary classification (expressed or not) or semiquantitative from 0 to 2 , 29 or 3In a consensus paper, the Society for Immunotherapy of Cancer has set a cut-off for TPS of ≥1% for carcinomas of the head and neck area . In thisFurthermore, controversies exist about the implemented antibodies to detect PD-L1 expression. In this context, a comparison of antibodies was found in two studies showing that the PD-L1 status can be influenced by the choice of assay , 39. ProBased on the results obtained in this study, PD-L1 expression was not seen a prognostic factor for OS and RFS. Neither a very high nor a very low level of PD-L1 had an influence on OS. Opposing statements can be found in the studies of Ahn et al. and Hanna et al. , 42. In Having this limitation in mind, one may hypothesize that a qualitative assessment of PD-L1 expression may be more efficient to detect possible responsiveness to immunotherapy in comparison to quantitative assessment. Here, a significant difference between the two sub-entities in the histoscore as a tool to assess these qualitative differences was found. This suggests that the OSCCs express PD-L1 with a higher intensity and tent to do so more frequently in comparison to OPSCC. Unfortunately, there are few comparative studies to compare these findings as the histoscore obtained by immunohistochemistry has not received much attention so far. Sadeghi et al. also used the H-score in their study on CD44 in colorectal cancer and divided their collective into two subgroups based on the mean value . Others The epidemiological characteristic of the investigated study cohort showed frequent localizations consistent with the literature: the tongue as the primary anatomical region for OSCC  and the th TNM classification has not yet been applied consequently. Next, an unusual high number of early stage tumors were included in this study that may have an impact on survival statistics in comparison to other studies. Methodologically, IHC staining was evaluated using a software, but the program had to be adapted to the tumor by a blinded pathologist so that complete objectivity cannot be given. In addition, the HPV association was detected with p16 staining which is used as the IHC gold standard. However, it is only considered as a surrogate marker with certain shortcomings in comparison to PCR detection of the specific HPV phenotype [There are certain shortcomings in this study. First and overall, the retrospective manner of this study may implicate a recall bias. As a strong example, the patients record were gathered from 2011 to 2017 and the 8henotype . Lastly,henotype . These fIn this study, no influence of PD-L1 expression on OS could be found. It was noticeable that the PD-L1 expression was 17.7% higher in OSCC than in OPSCC. This observation was not significant. A relationship between HPV and PD-L1 has not been established. It was found that OPSCC are significantly more likely to be p16+ than tumors of the oral cavity. Further studies are needed to investigate the role of PD-L1 in HNSCC.S1 Table(PDF)Click here for additional data file.


Objectives: Soft and hard tissue breakdown was histologically and radiologically assessed around implants with alternate, consecutively placed ligatures on the same edentulous dog hemimandible. The influence of ligatured implants (LI) on adjacent non-ligatured implants  was also evaluated. Material and Methods: Three months after tooth extraction, five dental implants were placed in the dog hemimandible. Two months after abutment placement, ligatures were placed subsequently two months apart on alternate implants, while both intermediate implants were left without ligatures. Ligatures were kept in place during the entire experiment, and no plaque control measures were taken. Eleven months post-implantation, the animal was sacrificed. Undecalcified ground sections were cut, stained with Masson Goldner and MOVAT Pentachrome and evaluated by light microscopy. Soft and hard tissue loss was assessed using histomorphometric and CBCT parameters. Results: All NLI presented deep false peri-implant pockets on the oral aspect and pronounced vertical bone resorption on the buccal aspect. After 2, 4 and 6 months, during the breakdown period, more than 30% of the bone was lost in LI in all directions, while, despite immediate vicinity, NLI displayed less destruction. Intense inflammation, typical for induced peri-implantitis, was present, with similar intensity in LI as NLI, but in different parts of the lesions. Morphometry confirmed intense soft tissue inflammation, more bone resorption and higher amounts of infiltrated connective tissue in LI when compared with NLI. Conclusion: Within the limits of the present pilot study, the adequacy of the experimental dog model based on ligature-induced peri-implantitis was able to be successfully challenged by non-ligature models of spontaneously occurring peri-implant inflammation, while meeting the requirements for experimental designs with a very small numbers of animals. The influence of implants with severe peri-implantitis on adjacent implants resulted in less than expected tissue loss in the latter accession numbers.  Peri-implantitis is an infectious disease and is considered as a common biological complication in implant therapy ,2,3,4,5.However, the EU regulations regarding the protection of the animals used for scientific research reflect an increasing determination to reduce the number of experimental animals and their pain. If death is an endpoint of the experiments, as few animals as possible should be used . Such reA new approach to the ligature model in dogs, which allowed the observation that the spontaneous progression of peri-implantitis may occur after the removal of ligatures, was introduced by Zitzmann et al.  and was More recently, it was suggested that the ideal canine peri-implantitis induction model would be a naturally occurring peri-implantitis induction, without the action of any ligature . The litSite-specific diseases, like peri-implantitis, are often attributable to local predisposing factors. Local contributors that can cause plaque-associated peri-implantitis include, among others, soft and hard tissue characteristics, surgical procedures, prosthetic factors and local precipitant factors  ,30,31. LAn unanswered question remains whether implants affected by peri-implantitis influence adjacent implants. Furthermore, in an experimental design, the extent to which ligature-induced peri-implantitis mimics a clinical situation may influence their non-ligated neighboring implant and even provoke peri-implantitis still remains unknown.Cone-beam computed tomography (CBCT) has been stated to be advantageous for the diagnosis of peri-implantitis defects  because As the peri-implantitis problem remains today largely unresolved, emerging new therapies try to address the disease; however, in order to make their clinical application efficient, validated experimental models that closely reproduce the pathophysiology of human peri-implantitis must be designed. From this perspective, the principal aim of this pilot study was the histomorphometrical and CBCT assessment of tissue loss progression in peri-implantitis induced by ligatures placed consecutively on alternate implants on the same edentulous dog hemimandible. The influence of ligatured implants on adjacent non-ligatured implants was also evaluated.To minimize the number of experimental animals to be sacrificed, this pilot study was conducted in a single animal, so that a maximum of data could be obtained from the experimental combination of implant positions and ligature placement.The endosseous dental implants  consisted of 5 titanium cylinders , etched homogenous implant surface from titanium grade 4 with a titanium plasma spray coating applied under vacuum conditions and a 1 mm highly polished collar. Highly polished titanium abutments were connected 3 months after insertion. The dog was housed under good general conditions in a single kennel with indoor and outdoor areas. The room temperature range was approximately 18 °C, with a humidity above 30%. The dog was fed once a day using a granulated dog food and water ad libitum. The maxilla was intact, without any general occlusal trauma, oral viral or fungal lesions. Clinical examination determined that the dog was in good general health, with no systemic involvement ,42.The experiment was performed on one ten-years old adult half-breed dog, with a fully erupted permanent dentition . All operative procedures were carried out under general anesthesia . To maintain hydration, the animal received a constant-rate infusion of Ringer’s solution while anesthetized. The protocol of the whole experiment was approved by the Ethical Commission of Scientific Research of UMFVBT . The experiment was finalized in December 2019. According to the timeline of the experiment , the preTo be used in further research, intravital polyfluorochrome labeling was carried out for the histological evaluation of the bone reactions through the intraperitoneal injection of oxytetracycline , 50 mg/kg body weight, every tenth day, from implant placement until abutment installation, and alizarin red S 1% , 40 mg/kg body weight, 10 days before sacrifice.Implants were exposed 3 months post-implantation and their healing abutments were connected . The flaTo check the peri-implant vertical alveolar bone status on the right side of the lower jaw at baseline, a conventional dental X-rays was taken immediately after the titanium abutments were installed, 3 months post-implantation .At the end of the experiment, 8 months after abutments were installed, the dog was sacrificed under general anesthesia . The jaw was sectioned at the midline, a segment of mandible, including the implants and ligatures in situ (8 cm × 4 cm), was separated and fixated in 10% neutral buffered formalin until laboratory processing. The entire segment underwent CBCT analysis. ® 3Dx, SOREDEX, Tuusula, Finland). The exposure settings were 12 mA and 89 kVp, with an acquisition time of 6.1 s. The scanning parameters were set to a volume size of 80 mm height and 80 mm width, a cross interval of 0.2 mm and a panorama interval of 0.3 mm. The measurements were performed under standardized conditions using the imaging software version OnDemand3D Dental 1.0.10.7462. The jaw segment underwent CBCT evaluation . The following landmarks were identified in each sagittal image at all buccal, oral, mesial and distal aspects: IS (implant shoulder), the bottom of the bone defect (BD) and the alveolar bone crest (BC) . At bothThe angulation of the peri-implant defects, as an additional measure of the extension of the bone destruction, was evaluated as well at the buccal, oral, mesial and distal aspects of the implants and represented the radiographic angle between the IS-BD and BD-BC segments. Technovit 9100  embedding resin was used for the infiltration and embedding steps. Sections were prepared using the cutting/grinding method described by Donath and Breuner  on a cutThe stained thin sections were scanned on a Zeiss Axio Scan Z.1 system  at 20× magnification. These images were used for morphometric measurements using the Icy Bioimage Analysis software v2.4 . All secThe following histomorphometric landmarks were identified in both the buccal and lingual aspects in each implant : implantLinear measurements of both the buccal and lingual aspects of each implant were made between: the implant surface (imp) and the alveolar bone crest (imp-BC); the implant shoulder (IS) and the level of the bone crest (BC) (IS-BC); the implant shoulder (IS) and bottom of the bone defect (BD) (IS-BD); the bone crest (BC) and the bottom of the bone defect (BD) (BC-BD); the level of the bone crest (BC) and the apical extension of the infiltrated connective tissue (aICT) (BC-aICT); the margin of the peri-implant mucosa (PM) and the level of the bone crest (BC) (PM-BC); the margin of the peri-implant mucosa (PM) and the implant shoulder (IS) (PM-IS); the margin of the peri-implant mucosa (PM) and the apical cells of the junctional epithelium (aJE) (PM-aJE); the margin of the peri-implant mucosa (PM) and bottom of the bone defect (BD) (PM-BD); the margin of the peri-implant mucosa (PM) and the apical extension of the infiltrated connective tissue (aICT) (PM-aICT); the apical termination of the junctional epithelium (aJE) and bottom of the bone defect (BD) (aJE-BD); and the most apical extension of the submarginal biofilm that was interposed between the implant and the pocket epithelium of the peri-implant mucosa (aPlaque) and bottom of the bone defect (BD) (aPlaque-BD).The following angles, measuring the buccal and oral extension of the bone destruction, were defined: the angle between the IS-BD and BD-BC segments on the buccal aspect and the angle between the IS-BD and BD-BC segments on the oral aspect. The area of the infiltrated connective tissue (ICT) was identified and traced using a mouse cursor.p-value of less than 0.05 was considered statistically significant.Using the implant as the statistical unit, the mean values and standard deviations were calculated separately for ligated implants (LI) and non-ligated implants (NLI). Histological measurements were performed on buccal and oral aspects for each implant and CBCT measurements were performed on the buccal, oral, mesial and distal aspect of each implant. The data rows were examined using the Shapiro–Wilk test for normal distribution. Due to the small sample size, a Wilcoxon test was performed only to compare the differences between the CBCT and histomorphometric results. A p = 0.277 > 0.05) for global measurements.During the experimental peri-implantitis, none of the five implants failed. Heavy plaque accumulation was observed around all healing abutments, and the peri-implant tissues became inflamed and bled, while implant No. 3 exhibited suppuration. After 2, 4 and 6 months, during the phase of experimental peri-implantitis (“breakdown period”), more than 30% of the bone was lost in LI, as shown by the CBCT images. However, NLI displayed less bone loss according to the design of the experiment. The Wilcoxon test for the differences between the histometric measurements with CBCT correspondents  was not statistically significant  was 1.70 ± 0.56 mm for LI and 0.39 ± 0.38 mm for NLI, describing, as expected, a wider bone destruction in LI. The corresponding mean vertical distance between the implant shoulder and the level of the bone crest (IS-BC) was 1.78 ± 1.31 mm for LI and 0.71 ± 0.63 mm for NLI, meaning that LI also exhibited more vertical bone loss. The mean distance between the implant shoulder and the bottom of the bone defect (IS-BD) was 3.12 ± 0.58 mm for LI and 0.79 ± 0.55 mm for NLI, illustrating, once again, an advanced bone loss around LI. Similar mean distances for IS-BD have been found in buccal and oral aspects on LI .The mean distance between the bone crest and the bottom of the bone defect (BC-BD) was 1.33 ± 0.85 mm for LI and 0.33 ± 0.39 mm for NLI. 2 for LI and 1.2 ± 0.62 mm2 for NLI . The mean value of the angle between the IS-BD and BD-BC segments was identical-51.71° ± 11.12 for LI and 51.75° ± 11.32 for NLI, despite the fact that in NLI the length of the buccal segment BC-BD was extremely reduced because of the severe resorption of the buccal cortical. Several distances between bony landmarks and soft tissue modifications have been noted. The mean distance between the bone crest and the apical extension of the infiltrated connective tissue (BC-aICT) measured 1.53 ± 0.91 mm for LI and 0.60 ± 0.34 mm for NLI, describing for the LI a larger area of bone that is in contact with infiltrated connective tissue. The mean distance between the margin of the peri-implant mucosa and the bottom of the bone defect (PM-BD) was 3.30 ± 1.37 mm for LI and 2.14 ± 0.37 mm for NLI, resulting in an increase of 65% in LI when compared to NLI. The mean height of the peri-implant mucosa in relation to the bone crest (PM-BC) was 1.96 ± 1.32 mm for LI and 1.80 ± 0.56 mm for NLI. The mean vertical distance between the margin of the peri-implant mucosa and the implant shoulder  was 0.18 ± 1.38 mm for the LI and 1.34 ± 0.60 mm for the NLI. However, these values have to be interpreted taking into account both „true” mucosal recessions  and mucosal enlargements, resulting in false peri-implant pockets with positive values. In this regard, it is to be noted that NLI displayed only false pockets, thus positive values. The mean length of the barrier epithelium (PM-aJE) was on the average 1.66 ± 0.96 mm for LI and 1.05 ± 0.61 mm for NLI (approximately 63% higher in LI), in contradiction with the expected pathophysiology of the peri-implant lesion. The ICT mean apical extension was 3.49 ± 1.41 mm in LI compared to 2.30 ± 0.90 mm for NLI (PM-aICT), representing 66% more soft-tissue surrounding the implants in LI, due to heavily infiltrated connective tissue. The connective tissue located between the epithelium and the bone defect  had a mean height of 1.63 ± 2.03 mm for LI and 1.08 ± 0.53 mm for NLI, being obviously more abundant in LI. Finally, the distance between the most apical extension of the submarginal biofilm (interposed between the implant and the pocket epithelium) and the bottom of the bone defect (aPlaque-BD) measured 2.24 ± 0.61 mm for LI and 2.43 ± 0.23 mm for NLI. The mean value of the area occupied by the infiltrated connective tissue was 3.51 ± 2.49 mmBeam-hardening artifacts, dark streaks between implants and the photon starvation effect were only observed occasionally. These adverse effects and the buccal bone resorption were associated with a difficult depiction of the landmarks. In our experimental model, the mean values of buccal, oral, mesial and distal CBCT measurements were used to comparatively assess the bone loss between LI and NLI .When comparing LI and NLI on all four aspects, the supra-crestal component of the defect (SC) was on the average 2.32 ± 1.03 mm for LI and 1.09 ± 1.07 mm for NLI, suggesting that more bone was lost around ligated implants. The intrabony component of the defect (IC) measured 1.46 ± 1.00 mm for LI and 0.29 ± 0.29 mm for NLI, illustrating a crater-like defect around LI. The mean dimension of the defect width (DW) was 1.53 ± 0.84 mm for LI and 0.33 ± 0.32 mm for NLI describing a wider defect for LI. Finally, the mean of the angle between the IS-BD and BD-BC segments measured on all four aspects was 42.23° ± 21.95 for LI and 33.43° ± 28.62 for NLI.A separate analysis of the mean values of mesial and distal CBCT measurements was effectuated for LI and NLI . It is sMoreover, a separate analysis of the mean values of buccal and oral CBCT measurements was effectuated for LI and NLI . Most ofOur experimental setting tried to reproduce the clinical reality of several adjacent similar implants but with different magnitudes of peri-implant involvement, while acknowledging the distance between the implants. The current study presents preliminary data aimed to confirm the influence of ligatured implants on adjacent non-ligatured implants, the latter presenting with naturally occurring mucositis and subsequent peri-implantitis as well—a situation encountered in numerous clinical situations. The study uses an animal dog model to study the experimentally induced peri-implantitis. For this purpose, the dog model was originally advocated by several authors ,23,45, sIn the above-mentioned study models, ligatures of cotton, silk or metal  were plaA recent study found that the number of adjacent implants is associated in humans with peri-implant disease, and this may be attributed to difficulties in performing adequate oral hygiene around multi-unit or splinted restorations, typically used in cases with multiple implants per patient . This isIn our study, the histological examination of peri-implantitis sites disclosed that the lesion occurred not only in the mucosa but also frequently extended into the marrow space of the surrounding bone tissue, in line with previous studies . The larThe ligature-induced marginal bone loss observed in this study is in agreement with data previously reported in a study that obtained similar results in the first two months after ligature placement . Although implants 1, 3 and 5 were ligated at different points, similar results were noted. This means that, in our study, a similar pattern and speed of bone resorption occurred at the ligated implants. Although the mentioned mean results were similar, the difference was seen on the buccal aspect. This observation seems to be in accordance with Monje et al. , affirmiIn both NLI implants, it was noted that the buccal plate underwent an almost complete resorption, up to the point that the segment BD-BC, which forms the angle between IS-BD and BC-BD, was reduced to less than 0.5 mm, making this particular angle measurable only under high magnifications. The above-mentioned buccal bone loss is in agreement with the CBCT observations of Monje et al., both on experimental peri-implantitis in dogs  and on hIn their experiment, Martins et al.  allowed The mean distance between the margin of the peri-implant mucosa and bottom of the bone defect (PM-BD) resulted in an increase of 65% in LI when compared to NLI, suggesting that an intense inflammation occurred in a short period of time, so that tissues did not have time to remodulate. The mean height of the peri-implant mucosa in relation to the bone crest (PM-BC) was 1.96 ± 1.32 mm for LI and 1.80 ± 0.56 mm for NLI, possibly suggesting that the height of the peri-implant mucosa remains constant although the bone support is progressively being lost. Finally, the distance measured between the most apical extension of the submarginal biofilm (interposed between the implant and the pocket epithelium) and the bottom of the bone defect (aPlaque-BD), apparently was maintaining a constant distance between the apical extension of the plaque and the bottom of the bone defect, despite the presence of ligatures as a mean of accumulating plaque.The histometric measurements with CBCT correspondents are SC, IC, DW, the angle made by IS-BD and BD-BC, all of them measured on buccal and oral. The Wilcoxon test showed that this difference was not statistically significant for global measurements. These results are in agreement with previous research that closely correlated CBCT analysis with the corresponding histological data . The mesThe present pilot study had several limitations. One of them is the number of implants used, limited to a single experimental animal, in accordance with the most recent animal ethical recommendations. The sample size, although small, is normal for a pilot study, respecting the recent ARRIVE guidelines. Recent animal model studies reflect this tendency as well . AnotherWithin the limits of the present pilot study, the adequacy of the experimental dog model based on ligature-induced peri-implantitis was able to be successfully challenged by the non-ligature models of spontaneously occurring peri-implant inflammation, while meeting the requirements for experimental designs with a very small numbers of animals. Using the same study design, the evaluation of the influence of implants with severe peri-implantitis on adjacent implants revealed less than the expected tissue loss in the latter, with CBCT data complementing the histomorphometry. This finding might influence the choice of therapeutical approach when dealing with the various degrees of severity of the disease on adjacent implants.


Plasmodium falciparum, scarce data exist regarding the association of P. vivax and HIV. In this cohort study, we assessed the effect of HIV on the risk of vivax malaria infection and recurrence during a 4-year follow-up period in an endemic area of the Brazilian Amazon. For the purpose of this study, we obtained clinical information from January 2012 to December 2016 from two databases. HIV screening data were acquired from the clinical information system at the tropical hospital Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD). The National Malaria Surveillance database  was utilized to identify malaria infections during a 4-year follow-up period after diagnosis of HIV. Both datasets were combined via data linkage. Between 2012 and 2016, a total of 42,121 people were screened for HIV, with 1569 testing positive (3.7%). Out of all the patients diagnosed with HIV, 198 had at least one episode of P. vivax malaria in the follow-up. In the HIV-negative group, 711 participants had at least one P. vivax malaria episode. When comparing both groups, HIV patients had a 6.48  (adjusted relative risk) greater chance of acquiring P. vivax malaria. Moreover, being of the male gender , Amerindian ethnicity , and a resident in a municipality of the Metropolitan region of Manaus  were independent risk factors associated with an increased risk of clinical malaria. Education ≥ 8 years  and living in the urban area  were associated to a lower risk of P. vivax malaria. A total of 28 (14.1%) and 180 (25.3%) recurrences  were reported in the HIV-positive and HIV-negative groups, respectively. After adjusting for sex and education, HIV-positive status was associated with a tendency towards protection from P. vivax malaria recurrences . HIV status was not associated with hospitalizations due to P. vivax malaria. CD4 + counts and viral load were not associated with recurrences of P. vivax malaria. No significant differences were found in the distribution of parasitemia between HIV-negative and HIV-positive P. vivax malaria patients. Our results suggest that HIV-positive status is a risk factor for vivax malaria infection, which represents an additional challenge that should be addressed during elimination efforts.Globally, malaria and human immunodeficiency virus (HIV) are both independently associated with a massive burden of disease and death. While their co-infection has been well studied for  This overlap favors Plasmodium-HIV co-infections, which are responsible for deaths of millions of individuals each year4. Although the consequences of co-infection with HIV-Plasmodium are not fully understood, the available evidence suggests that infections act synergistically and together may result in worse clinical outcomes5. Recent studies show that people living with HIV have more episodes of severe and lethal P. falciparum malaria, and more malaria treatment failures9. HIV can increase parasitic loads in malaria patients and consequently increase malaria transmission rates12. Moreover, P. falciparum infection may increase HIV plasma viral load and reduce TCD4 + cells14; this, in turn, suggests that malaria can lead to a faster progression from HIV infection to AIDS and increased risk of therapeutic failure after antimalarial chemotherapy15. However, the evidence on HIV-Plasmodium co-infection is mostly restricted to P. falciparum endemic areas in sub-Saharan Africa where the two diseases have the highest burden16.Malaria and HIV/AIDS are major public health problems and globally have a great geographical overlap, with most affected people living in sub-Saharan Africa, the Indian subcontinent and Southeast Asia, Latin America, and the CaribbeanP. falciparum malaria is unknown, and further research should be conducted in other settings to assess the interaction of HIV infection with other Plasmodium species, such as P. vivax17. Globally, ~ 229 million malaria cases were reported in 2019, with at least 6.9 million cases due to P. vivax (3%)18. Despite its comparatively lower numbers, P. vivax is geographically the most widely distributed cause of malaria, with its highest endemicity found in Southeast Asia, Central Asia, and Latin America, and a much less frequent presence in Africa19. P. vivax is a challenging parasite to eliminate due to its peculiar biology, which causes the different origins of a P. vivax malaria episode that include acquired infection by mosquito bites, relapse from liver-stage hypnozoites, and recrudescence from blood-stage treatment failure20. Clinically, P. vivax infection ranges from asymptomatic individuals carrying low parasitemia to severe disease and death21. Reports of HIV-P. vivax co-infection are scarce in the literature, with only a few studies describing their clinical outcomes27. In one study in the Brazilian Amazon, of 21 patients with HIV-P. vivax co-infection hospitalized in a referral hospital, severe malaria was diagnosed in 5 (23.8%), and one patient died (4.8%)27.The impact of HIV on susceptibility to non-falciparum malaria among HIV-infected patients, research on co-infection with the most widely distributed vivax malaria has so far been neglected. In this study, we evaluated the impact of HIV infection on P. vivax malaria incidence and the frequency of recurrences in a malaria endemic area of the Brazilian Amazon. The hypothesis of this study was that HIV infection may increase P. vivax malaria susceptibility and recurrence rates.Although over the past three decades, there has been clear evidence of an association between the increased risk of Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD) between January 2012 and December 2016. After removing duplicates for individuals tested more than once, 42,121 were included in the study, with 1569 being classified as HIV- positive (3.7%) and 40,552 as HIV-negative (96.3%)  and a younger mean age (P < 0.001). Regarding municipality of residence, HIV-positive status was less prevalent in Manaus compared to the municipalities of the metropolitan region (P = 0.046) ; P < 0.0001], male gender , Amerindian ethnicity , and residence in a municipality in the Metropolitan region of Manaus  were independently associated with an increased risk of P. vivax malaria. Education ≥ 8 years  and living in the urban area  were independently associated to a decreased risk of P. vivax malaria.Table P. vivax malaria . After adjusting for sex and education, the association between HIV-positive status with protection from recurrences was not detected   and 180 25.3%) recurrences were reported in the HIV-positive and HIV-negative groups, respectively. In the univariate analysis, HIV-positive status was associated with protection from recurrences of % recurreP. vivax malaria were documented in the HIV-positive group (2.7%) and 40 in the HIV-negative group (4.5%) . No deaths from malaria were reported.Six hospitalizations due to P. vivax malaria. Individuals carrying a viral load that was < 50 copies/mL at baseline presented a higher risk of malaria  and P. vivax malaria  compared to those with a viral load > 100,000 copies/mL, after adjusting by gender, ethnicity, municipality of residence, years of education and zone of residence   of the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado . The ERB gave a waiver for informed consent. After database linkages, the final dataset was anonymized before statistical analysis.P. vivax infections28. The national HIV prevalence is estimated at 0.4%, with a noticeably higher HIV incidence rate of 34.8/100,000 population in the Brazilian Amazon, compared to the overall national rate of 17.8/100,00029.Out of all malaria cases in Brazil, more than 99% occur in the Brazilian Amazon region, of which about 89% are attributable to FMT-HVD is a tertiary, referral healthcare center for infectious diseases in Manaus, state of Amazonas, western Brazilian Amazon and, in its outpatient clinic and emergency department, it receives all patients seeking care for infectious and parasitic diseases or those referred from other health units in the city of Manaus and the surrounding areas. HIV testing is provided after careful health screening for possible epidemiological risk and exposure. Diagnosis and treatment are free of charge. For both diseases, any positive HIV or malaria diagnoses are compulsorily recorded in structured forms available on-line as part of the Brazilian Ministry of Health's notification system. FMT-FVD is responsible for the follow-up and management of ~ 80% of the HIV/AIDS cases in the state of Amazonas.30, as reported on the FMT-HVD Information System (iDoctor).This is a concurrent, cohort study, based on surveillance data from patients tested for HIV at FMT-HVD and malaria diagnosis data and case recurrences recorded in the National Malaria Epidemiological Surveillance Information System  between January 2012 and December 2016. In this study, exposure was defined as a confirmed HIV-positive status, according to the national HIV infection diagnosis guidelineBio-Manguinhos, Fiocruz) with 100% sensitivity and 99.8% specificity31 was used in mobile testing campaigns and at the FMT-HVD outpatient clinics. For each positive test, blood samples were collected at the FMT-HVD laboratory, and the western blot technique was used to confirm the diagnosis, and the patient was then referred to the HIV ward for consultations, psychological support and ART therapy. All the remaining individuals with HIV-negative test results were included as non-exposed controls. HIV-positive patients were stratified by CD4 cell counts (/mm3) and viral load (copies/mL), via data gathered from the FMT-HVD SISCEL (Laboratory Test Control System) database. The predictor variables sex, age, education (in years of schooling), self-reported ethnicity, place of residence , and municipality of residence were also used. All HIV-positive patients were treated according to the Brazilian Ministry of Health Guidelines32.In short, a rapid diagnostic test (RDT)  P. falciparum infection, (iv) P. vivax malaria recurrences up to 90, 180 days and 4-years, (v) malaria-associated hospitalizations, (vi) malaria-associated deaths, in a 4-year follow-up, and (vii) parasitemia. Recurrence was defined as the occurrence of at least one additional malaria infection during the follow-up period. Furthermore, the study aimed to determine any association between age, sex or parasitemia as a potential additional risk factor for malaria reinfection. In Brazil, thick blood smears (TBS) are routinely performed for the diagnosis of malaria, and are prepared according to the Walker technique33 and then evaluated by a local microscopist. The results of peripheral parasitemia are given using the following semi-quantitative system: < 1/2 + (< 200 parasites/mm3); 1/2 + (200–300 parasites/mm3); 1 + (301–500 parasites/mm3); 2 + ; 3 + ; and 4 + . All positive slides and 10% of negative slides are routinely reviewed at a referral unit with experienced microscopists. In case of divergence, the reviewed result is updated on the on-line system. Only participants with a positive TBS for Plasmodium spp. are treated according to the Brazilian Anti-Malarial Treatment Guidelines34. The malaria patients' data are entered into the SIVEP Malaria  database. Data on malaria-associated hospitalizations and deaths were extracted from HIS  and MIS  databases, respectively.The present study was designed to determine the risk of (i) malaria infection, (ii) 35 and was installed on a Linux Workstation, Intel Core i7-8700, with 32 GB of RAM and 12 physical processing cores. In the absence of a unique identifier, Tucuxi-BLAST used the patient's name, date of birth and mother's name. Malaria episodes reported before the participants' entries were then excluded from the generated database. All remaining matches were double-checked and false matches were also excluded. This inspection included visual confirmation of homonyms, possible siblings (twins), and duplicity. Finally, the list of malaria cases obtained was linked to the HIS  and MIS  databases to investigate these outcomes. We obtained a final selection of pairs identified as likely to be from the same patients by automatic verification, applying a probability threshold (probability > 0.7) for all linkages. A final identification (ID) was created for all included participants.Duplicated HIV tests were excluded from the iDoctor database  and, in the case of individuals that had had more than one HIV test in the period, the first positive result was considered. Patients exposed to disease (HIV-positive) and the controls (HIV-negative) were included in the study according to the day of confirmation of HIV-status. Patients entered and left the study at different points in time. The HIV-status database and the SIVEP-malaria database were both deduplicated and further the record linkage was performed  of Tucuxi-BLAST software, which uses the DNA-encoded approach2) test were used to compare proportions, as appropriate. Crude relative risk (RR) with its respective 95% confidence interval (95% CI) was determined in a univariate analysis. Logistic regression was used for the multivariate analyses and the adjusted RR (ARR) with 95% CI were also estimated. A log binomial multivariate generalized linear regression was performed using an automated backward and forward stepwise estimation. All variables that were associated with dependent variables at a significance level of P < 0.200 in the univariate analysis were included in the multivariate analysis. Statistical significance was considered when P < 0.05 in the Hosmer–Lemeshow goodness-of-fit test. Kaplan–Meier survival estimates were used to compare recurrences over follow-up time. Additionally, a survival analysis for recurrent events was performed36. A two-tailed P < 0.05 was considered significant. The statistical analyses were carried out using R software (version 4.1.0), R Studio (version 1.4.17), and Stata v.13.0 .Descriptive statistics were used for demographic variables. Student's t test was used to compare means while Fisher's exact or the Chi-squared (XSupplementary Information.